The biosynthesis, mechanistic enzymology and molecular genetics of terpenoid biosynthesis will be investigated using a wide variety of chemical, biochemical, structural biological, and bioinformatic tools. The focus will be on a family of microbial sesquiterpene (fifteen-carbon terpenoid) synthases that catalyze the cyclization of the universal precursor farnesyl diphosphate to a large number of cyclic sesquiterpenes, including pentalenene, germacradienol, and the newly discovered compound epi-isozizaene, as well as the metabolic conversion of these compounds to metabolites such as the antibiotic pentalenolactone or the volatile organic geosmin, the odoriferous constituent of Streptomyces and of blue-green algae that is responsible for the characteristic earthy smell of freshly turned soil as well as the undesirable off-taste associated with contaminated drinking water and foods. We will also mine microbial genome databases for identification, biochemical characterization, and mechanistic investigation of new terpenoid synthases and downstream biosynthetic enzymes from bacterial and fungal sources. In order to explore terpenoid mechanism and structure space as broadly as possible, we have devised a set of mutually complementary experimental approaches involving: A. Mechanistic studies of terpene synthases to define the mechanism of formation of individual sesquiterpenes from FPP. These studies will include site-directed mutagenesis of terpene synthases, exploiting the tendency of such mutants to produce mixtures of aberrant products that are diagnostic of the normally cryptic intermediates of the carbocationic cyclization cascade. Isotopically sensitive branching will be used to establish the order in which bonds are made and broken and elucidate the nature of key carbocationic intermediates. Our studies will address three central issues in terpene biosynthesis: 1) How does a cyclase impose a specific conformation on its acyclic substrate FPP and derived intermediates? 2) How does a cyclase manage positively charged intermediates, including catalysis of the initial ionization of the substrate, through stabilization of cationic intermediates, to termination of the reaction by quenching of positive charge? and 3) What are the relationships among terpene synthase sequence, structure, and function? B. Isolation, expression and mechanistic investigation of new terpenoid synthases as well as downstream biosynthetic enzymes from bacterial and fungal sources, identified by bioinformatic analysis of genomic sequences. C. Determination of the X-ray crystallographic structures of wild-type and mutant terpenoid synthases, both substrate-free and with bound substrate and intermediate analogs, in collaboration with Prof. David W. Christianson (University of Pennsylvania), as well as determination of the structures of selected downstream biosynthetic enzymes in collaboration with Prof. Gerwald Jogl (Brown University.) Relevance Terpenoid metabolites, the largest known group of natural products, include hormones, antibiotics, anti- tumor agents, immunosuppresants, neurotoxins, flavor and odor constituents, and pigments, among a wealth of medicinally, physiologically, or commercially important properties. Studies of the biosynthesis of these compounds by bacteria and fungi can not only lead to an understanding of fundamental metabolic pathways, but facilitate the development of practical tools for the generation of new medicinal agents or for prevention of the formation of toxic or otherwise undesirable metabolites by pathogenic or environmental microorganisms. Finally, the use of genome mining to biochemically characterize genes of unknown function will lead to fundamental new biological insights into the central issue of modern molecular biochemistry, the relationship between protein sequence, structure, and function.